| Spodoptera exigua(Hübner) is an important world-wide spread polyphagous pest. The wild-type laboratory strain(named SEW) of S. exigua was established with a collection from cotton fields in Jingzhou, Hubei in 2003. At the sixth generation, some black pupae spontaneously occurred within the SEW strain. The adults that emerged from the black pupae were crossed to establish the pupal melanic strain(named SEM). The black pupa phenotype is very stable and never changed since the SEM strain established. This study included observation of the pigmentation process of the pupa from both strain, comparison of the fitness of the two strain, analysis of the different expression pattern of melanin synthesis genes between the two strains and their transcription regulation mechanism. This study provide theoretical basis for explaining the mechanism underlined the pupal melanic mutant and the relationship between the pupal melanic mutation and fitness change. The results were listed below in details:1 The SEM strain of S. exigua showed fitness-gain compared to the SEW strainObservation and photograpgy of the pigmentation process showed that the SEM strain were globally black at the pupa stage, and no pigmentation difference was observed in egg, larva and adult stages. After pupation, the melanic SEM pupae gradually accumulate melanin to become completely black within 6 hours. The melanic SEM strain exhibits faster development in all life stages, heavier pupa weight, higher mating rate, more mating time, higher fecundity, and accordingly, higher net reproductive rate(R0=275.66) and population trend index(I=123.73). The mating times per female of the reciprocal crosses(♀SEW×♂SEM: 2.40±1.30 times;♀SEM×♂SEW 2.43±1.22 times) and the SEM intracrosses(1.97±1.01 times) were significantly higher than those of the SEW intracrosses(1.44±0.62 times). This represents a rare case of melanization that has fitness gains, rather than costs. Analysis of the life-history traits of this case and 14 previously reported cases of insect melanism indicate that specific melanization origin, stage and variation type determines whether melanism will cause fitness gain or cost.2 The overexpression of TH played an important role in pupal melanism of S. exiguaPCR with degenerate primers was performed to amplify and clone the fragments of tyrosine hydroxylase(TH) gene, dopa decarboxylase(DDC) gene and Laccase 2 gene, and semi-quantitative RT-PCR was performed to compare the expression level of the three genes between the SEW strain and SEM strain. The results showed that the TH and DDC were both over expressed in the SEM strain compared to the SEW strain. The full length c DNA sequences of tyrosine hydroxylase(TH) and dopa decarboxylase(DDC) were cloned through RACE, and their temporal expression patterns in the integument were compared during the larval-pupal metamorphosis process of the S. exigua wild type(SEW) and melanic mutant(SEM) strains by q RT-PCR. No amino acid change in the protein sequence of TH and DDC was found between the two strains. TH expressed more abundantly than DDC gene in both SEW and SEM during larva-pupal process. TH significantly over-expressed(15.19-fold) in the integument of the SEM strain at late-prepupa compared with that of the SEW strain. DDC over-expressed in the SEM at both late-prepupa(32.44-fold) and 0h pupa(3.08-fold), respectively, compared with those of the SEW strain. RNAi through ds RNA injection was performed to knock down the TH gene and resulted in lighter color in the SEM pupa. Feeding the fifth instar larvae of SEM with diets incorporated with 1 mg/g of the DDC inhibitor L-α-Methyl-DOPA and 0.75 mg/g of the TH inhibitor 3-iodo-tyrosine(3-IT) resulted in 20% pupae with partially-rescued phenotype and 68.2% of pupae with partially- or fully-rescued phenotype, respectively. These results indicate that overexpressions of TH and DDC are involved in the pupal melanization of S. exigua, and TH plays a more important role than DDC.3 Promoter activity of TH gene was under complex regulationThree 2.38 kb length promoter region unpstream of the transcription initiation site of TH gene were cloned from the US wild-type, the SEW strain and the SEM strain of S. exigua through Genome Walking, respectively. The cis-acting elements in the known TH promoter sequence were predicted with the JASPAR database. There were total 55 predicted cis-acting elements with 100% identity with the database in the TH promoter region, and they were belonged to 24 transcription factors. 7 βFTZ-f1 response element-like regions were found in the TH promoter region. Dual-luciferase assays were performed to test the activity of 5’progressive deleted TH promoter to figure out the essential region and the regulation region in the promoter.4 βFTZ-f1 over-expressed in the SEM strainThe full-length c DNAs of the βFTZ-f1 gene from the US wild-type, the SEW strain and the SEM strain of S. exigua were cloned through Genome Walking and 3’RACE. Semi-quantitative PCR and q RT-PCR were performed to compare the expression level of βFTZ-f1 gene in the integument of both the SEW strain and the SEM strain during larval-pupa process. Sequence alignment showed that there was one amino acid difference between the predicted amino acid sequences of the SEW strain and the SEM strain, and there were four amino acid differences between the predicted amino acid sequences of the SEM strain and the US wild-type. The peak of βFTZ-f1 gene expression occurred at the late-prepupa stage, and it over-expressed in the SEM strain(7.48-fold) compared to that of the SEW strain. It also over-expressed in the SEM strain(1.74-fold) at the 0h pupa stage compared to that of the SEW strain. These results indicated that over expression of βFTZ-f1 gene was probably involved in the pupal melanic mutation.5 The promoter activity of TH gene was up-regulated by both βFTZ-f1 and FTZThe eukaryotic expression vector and luciferase report vector were used to expression βFTZ-f1 and TH promoter, respectively, in Sf9 cells and He La cells. The activity of TH promoter was upregulated by the overexpression of βFTZ-f1 in Sf9 cells(2.66-fold) and He La cells(1.60-fold), respectively. Further, the coactivator of βFTZ-f1, FTZ, was confirmed to upregulate TH promoter activity(1.79-fold) in the Sf9 cells with a similar function with βFTZ-f1. EMSA assays were performed to confirm one FTZ binding site in the TH promoter region, but no βFTZ-f1 binding site was located. The deletion and mutation of this FTZ binding site did not affect the upregulation of TH promoter by the FTZ and βFTZ-f1. Finally, more 5’progressive deletion constructs of the TH promoter were made and the activity were tested with βFTZ-f1 overexpression, and the results showed that the region between-2200 ~-2160 unsptream of the TH transcription initiation site included the putative βFTZ-f1 response element.In conclusion, firstly, the SEM showed fitness gain with the pupal melanic phenotype, which is a rare case in the labrotory occurred melanic insect. Secondly, overexpression of TH gene played an important role in the pupal melanization. Thirdly, βFTZ-f1 and FTZ can upregulate the promoter activity of TH gene, and the putative βFTZ-f1 binding site could be a new one. |
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